COMPOSITIONS CONTAINING 3-NITROIMIDAZO(1,2-b)PYRIDAZINES AND METHOD OF USE FOR TREATING AMOEBAE AND TRICHOMONAE

ABSTRACT

Compositions of a pharmaceutically acceptable carrier and a 3nitroimidazo(1,2-b)pyridazine and methods of using the same are described. These compositions are useful for their anti-protozoal activity as anti-trichomonal and anti-amebic agents.

United States Patent Tomcufcik et a].

[ 1 Jan. 16, 1973 Inventors: Andrew Stephen Tomcufcik, Old

Tappan; Raymond George Wilkinson, Montuale, both of NJ.

Assignee: American Cyanamid Company,

Stamford, Conn.

Filed: Feb. 24, 1971 Appl. No.: 1 18,509

US. Cl ..424/250 Int. Cl. ..A6lk 27/00 Field of Search ..424/250 OTHERPUBLICATIONS Kobe et al. Tetrahedron Vol. 24, Jan. 1968, pages 239 &242.

Primary ExaminerSam Rosen Attorney-Ernest Y. Miller [57] ABSTRACTCompositions of a pharmaceutically acceptable carrier and a3-nitroimidazo[ l,2-b]pyridazine and methods of using the same aredescribed. These compositions are useful for their anti-protozoa]activity as antitrichomonal and anti-amebic agents.

6 Claims, No Drawings COMPOSITIONS CONTAINING 3- NITROIMIDAZOU,2-B)PYRIDAZINES AND METHOD OF USE FOR TREATING AMOEBAE AND TRICIIOMONAESUMMARY OF THE INVENTION This invention relates to compositionscontaining 3- nitroimidazo[l,2-b]pyridazines and methods of using thesame.

Compositions of this invention have, as the active component, compoundsillustrated by the following formula;

wherein R is selected from the group consisting of hydrogen and loweralkyl.

In the above compounds lower alkyl is intended to include those havingone to four carbon atoms.

The present compounds can be prepared by the following method. Thereaction of an imidazo[l,2-b] pyridazine with nitric and sulfuric acidwhich can be illustrated as follgws N NO R1 R1 \\N/ 2 wherein R is ashereinbefore described. The reaction can be carried out at roomtemperature. It is preferable to use about 70 percent nitric acid withabout 96 percent sulfuric acid. When the reaction is carried out aboveroom temperature then lower concentrations of acids are suitable.

The present compounds as active components are useful for theiranti-protozoal activity as antitrichomonal and anti-amebic agents inwarm-blooded animals. They may be used in dosages in the range of fromabout 0.5 mg. to 30 mg. per kilogram of warmblooded animal. They can beformulated and used in well known pharmaceutical forms such as pills,capsules, tablets, liquids, liquid filled capsules and the like. Thevarious excipients, fillers, flavoring agents, etc. used in preparingthe pharmaceutical forms are well known to those skilled in the art.

The amebicidal properties of the present compounds are measured by meansof an assay devised from W. R. Jones, The Experimental Infection of RatsWith Entamoeba histolytica and a method for Evaluating the Anti-AmoebicProperties of New Compounds, Annals of Tropical Medicine andParasitology, volume 40, pages 130-140 I946). The assay is carried outas follows:

The test organism is Entamoeba histolytic NIH 200 a. Cultures aremaintained on Cleveland Collier liver infusion medium with serum saline1:] overlay in 3 X 5 test tube slants. Rice powder is added as a growthfactor. Cultures are transferred at 5 day intervals and kept at 37 C. A48 hour culture is used for the test inoculum and harvested the morningof the test by collecting the sediment containing rice powder andamoebae found at the junction of the butt and the slant. The amoebae arecounted and the amount of inoculum for injection is adjusted to containapproximately 200,000 to 250,000 amoebae. Female Wistar strain albinorats from the Royalhart Farms weighing 20-35 grams are used. The cecumis exposed during laporatomy and the amoebaerich inoculum is injectedinto the anterior section. The incision is closed with autoclips.Procedures are sterile throughout the course of the surgery. Theinfected rats are divided randomly into groups of IO. Treatment is begunon the day of infection. Drugs are premixed in a standard laboratoryfeed such as Purina Lab. Chow sold by the Ralston Purina Company. Ratsare maintained on the drug diet for 5 days at the end of which they arenecropsied and the cecum examined both macroscopically for pathologicfeature of infection and microscopically for the presence of amoebae.Scores of one each are recorded for evidences of mucous, fibrosus, andlesions or inflammation. A score of one is recorded for a finding of1-20 amoeba and a score of two for a finding of more than 20 amoeba on astandard slide preparation. Total score of zero to five, thus, ispossible per rat at necropsy. The The arithmetic mean of the combinedA.D.I.s (average degree of infection) in a test or control group of ratsis considered to be the group ADI. Activities are expressed inpercentage of suppression of group ADI of a test group to the group ADIor a control group. Consumption of test compound is determined from theweight of feed consumed. The present compound 3-nitroimidazo-[ l,2-b]pyridazine has a minimum effective dose (60 percent supp. of controlA.D.I.) mg./kg./day X 5 of 30.

The present compounds have shown activity as trichomonicides in testsdesigned to detect this activity. One such test is carried out asfollows:

Female albino mice (Royalhart ICR strain) are inoculated subcutaneouslywith 50,000 to 100,000 Trichomonas vaginalis (Thorns strain) suspendedin a cysteine-peptone-liver infusion-maltose medium described by GarthJohnson and Ray E. Trussell, Experimental Basis for the Chemotherapy ofTrichomonas vaginalis Infections I., Proceedings of the Society forExperimental Biology and Medicine Volume 54, pages 245-249 (1943). Incontrol animals, approximately one week postinoculation, the site ofinoculation is marked by a subcutaneous abcess which contains numeroustrichomonads in a menstruum of pus. In effectively treated animals theabcesses are either undetectable or greatly reduced in size, the motiletrichomonads cannot be detected in the lesion-derived material afterprolonged microscopic examination. Presence of a single motiletrichomonad after treatment is recorded as a negative result.

Treatment by test drugs consists either of one or more oral dosessuspended in 0.2 percent agar and administered by gavage one day postinoculation, or by administration in the diet for 5 consecutive daysbeginning one day postinoculation. The diet is a commercial laboratoryfeed sold under the trademark Purina Lab. Chow by the Ralston-PurinaCompany. The test compound is mixed thoroughly in the carrier; 0.2percent agar, 0.5 percent carboxymethylcellulose or ground laboratoryfeed. Each regimen is administered to a test group consisting of five orten mice. Control groups of five or ten mice receive the carrier alone.Gavage doses are estimated for the average mouse weight obtained justbefore dosing. Drug intakes resulting from diet therapy are estimatedfrom average mouse weights and total group feed intakes during thetreatment period. Activities in this test compare to that of2-methyl-5-nitro-l-imidazolethanol, a well recognized trichomonicide.The following Table I shows the activity of the present compounds.

TABLE I The activity of the present compounds against subcutaneousTrichomanas vaginalis infections in mice No. cleared"-/No. treated aftersingle gavage dose, mgJkg.

Compounds 100 50 2.5 12.5 6.2

3-nitroimidazo- 10/10 l/l0 10/10 10/10 [l,2-b]pyridazine6-methyl-3-nitroimidazol1,2-b]- 10/10 pyridazine Number of mice in whichno motile trichomonads were detected microscopically, at autopsy dayspost treatment.

Compositions containing as the active component the3-nitroimidazo[1,2-b]pyridazines may be administered to warm-bloodedanimals orally, or parenterally if desired, and when so administered,may be considered as an agent for the therapeutically desirabletreatment of protozoal infections such as amebic or trichomonalinfections in daily doses ranging from about 0.5 mg. to about 30 mg. perkilogram. The dosage regimen can be adjusted to provide optimumtherapeutic response. Thus, for example, several smaller doses may beadministered daily, or the dose may be reduced or increasedproporitionately as indicated by the requirements or the particulartherapeutic situation.

For therapeutic administration the active components of this inventionmay be incorporated with pharmaceutically acceptable carriers such asexcipients and used, for example, in the form of tablets, dragees,capsules, suppositories, liquids, elixirs, emulsions, suspensions or thelike. Such comparisions and preparations should contain at least 0.1percent active component. The percentage in the compositions andpreparations, may of course, be varied, and may conveniently be between2 and 60 percent or more of the weight of the unit. The amount ofcompound in such therapeutically useful compositions or preparations issuch that a suitable dosage will be obtained. Preferred compositions orpreparations according to the present invention are prepared so that adosage unit form contains between about and about 300 milligrams of theactive compound. Obviously, in addition to the therapeutic compound,there may be present excipients, binders, fillers and othertherapeutically inert ingredients necessary in the formulation of thedesired pharmaceutical preparation.

SPECIFIC DESCRIPTION The following examples describe in detail thepreparation of representative compounds of this invention andformulations containing these compounds.

EXAMPLE'l Preparation of 3-Nitroimidazo[ 1,2-b]pyridazine To imiclazo[1,2-b1pyridazine (Kobe et al. Tetrahedron 24, 239 (1968) is added withstirring concentrated sulfuric acid and the mixture cooled to about 10C. Stirring is continued and during 5 minutes nitric acid is addeddropwise. The reaction mixture is stirred at room temperature for 20minutes and poured onto crushed ice. The separated product isrecrystallized from water and pale yellow needles are obtained, meltingpoint l99-20l C;

EXAMPLE 2 Preparation of 6-Methyl-3-nitroimidazo[ 1,2-

blpyridazine Five and five-tenths grams of -methylimidazcl 1,2-

b]-pyridizine is added to 18 ml. of concentrated sulfuric acid. Thesolution is stirred vigorously as 4.8 grams of solid potassium nitrateis added in small portions. When addition is complete, the reaction isheated at 90 C. for 40 minutes. It is then drowned into ice and water.After near neutralization with concentrated ammonium hydroxide, themixture is extracted with chloroform. The chloroform is removed underreduced pressure, leaving a yellow crystalline residue. This is purifiedby recrystallization from a mixture of chloroformand ethanol, yielding5.2 grams of pure compound, melting at 203-204 C.

Analysis: Calcd. for C H N,O .C, 47.19; H, 3.40; N, 31.45.

Found: C, 47.1 1; H, 3.50; N, 31.48.

EXAMPLE 3 Suppositories containing 3-nitroimidazo[ 1,2- b]pyridazine 30suppositories g.

3-nitromidazo[ l,2'blpyridazine 7.5 Purified water qs AD 20 Gelatingranular 40 Glycerin 140 EXAMPLE 4 Preparation of Hard Shell Capsulescontaining 3- nitroimidazo-[ l ,2-b]pyridazine per 1,000 Capsules g.

3-nitroimidazo[ 1,2-b1pyridazine 200.0 Lactose 900.0 Magnesium stearate10.0

The ingredients are blended together. The mixture is used to fill hardshell capsules of a suitable size each containing 200 mg. of activecomponent.

EXAMPLE 5 Preparation of Tablet Compositions Containing 6-methyl-3-nitro-imidazo[ l ,Z-blpyridazine per 1,000 Tablets g.6-rnethyl-3nitroimidazol l ,2-bl- 100.0 pyridazine Corn starch USP 300.0Dibasic calcium phosphate 2150.0 Magnesium stearate 600.0

N N N O z wherein R is selected from the group consisting of hydrogenand lower alkyl and a pharmaceutically acceptable carrier.

2. The method in accordance with claim 1, wherein thenitroimidazopyridazine is 3-nitroimidazo[ 1 ,2-b1- pyridazine.

3. The method in accordance with claim 1, wherein thenitroimidazopyridazine is 3-nitroimidazo[ l ,2-b]- pyridazine and theprotazoa is an amoebae.

4. The method in accordance with claim 1, wherein thenitroimidazopyridazine is 6-methyl-3-nit roimidazo[ l,2-b]pyridazine.

5. The method in accordance with claim 1, wherein thenitroimidazopyridazine is 6-methyl-3-nitroimidazo[],2-b1pyridazine andthe protozoa is a trichomonae.

6. A therapeutic composition useful for the control of protozoa selectedfrom the group consisting of amoebae and trichomonae in warm-bloodedanimals which comprises a pharmaceutically acceptable carrier and from10 to 300 mg. of a nitroimidazopyridazine of the formula:

wherein R is selected from the group consisting of hydrogen and loweralkyl.

2. The method in accordance with claim 1, wherein thenitroimidazopyridazine is 3-nitroimidazo(1,2-b)-pyridazine.
 3. Themethod in accordance with claim 1, wherein the nitroimidazopyridazine is3-nitroimidazo(1,2-b)-pyridazine and the protazoa is an amoebae.
 4. Themethod in accordance with claim 1, wherein the nitroimidazopyridazine is6-methyl-3-nitro-imidazo(1,2-b)pyridazine.
 5. The method in accordancewith claim 1, wherein the nitroimidazopyridazine is6-methyl-3-nitro-imidazo(1,2-b)pyridazine and the protozoa is atrichomonae.
 6. A therapeutic composition useful for the control ofprotozoa selected from the group consisting of amoebae and trichomonaein warm-blooded animals which comprises a pharmaceutically acceptablecarrier and from 10 to 300 mg. of a nitroimidazopyridazine of theformula: